Adenovirus E1b region-coded 19K tumor antigen plays an important role in cell transformation in cooperation with the E1a proteins. We propose to investigate the role of 19K in adenovirus-mediated transformation using genetic and biochemical approaches. It appears that E1a-mediated transformation may be negatively regulated by other early viral genes. We will investigate the effect of the 19K gene relieving the transformation inhibitory effect of other early genes by DNA transfection and by using viral double mutants. Physical association of 19K with other early proteins will be studied by coimmunoprecipitation analysis of proteins cross-linked in living cells. Other early genes that interact with 19K protein will also be investigated using revertants of a 19K mutant, cyt6. The site of reversion will be mapped by marker rescue and by DNA sequence analysis. One of our major goals would be to identify cellular factors that physically interact with 19K during transformation. These proteins will be identified by coimmunoprecipitation analysis of proteins cross-linked in living cells over-express the 19K protein. The identity of the cellular proteins will be determinied by partial sequence determination of the genes coding for these proteins. To probe the structure and function of 19K, mutants with one or two amino acid substitutions or deletions will be constructed in the regions which appear to be functionally important. We have noted that a region of the 19K protein is homologous to a region of polyoma middle T (mT) antigen which is important for trans- formation. We will study the functional similarity between this 19K protein region and the corresponding mT protein region by domain substitution. We have observed that 19K plays a role in calcium mobilization. We will determine whether 19K plays a role in signal transduction at the level of generation of second messengers by studying the turnover of phosphoinositotides. As 19K appears to alter the architecture of the endoplasmic reticulum, we will also investigate whether 19K plays a role in calcium release from the endoplasmic reticulum by using isolated ER membrane vesicles in the presence of inositol tris- phosohate (InsP3). The 19K protein contains an amphipathic acidic domain similar to the calcium regulating domain of epidermal growth factor receptor (EGFR) and related receptors. To determine whether the acidic domain of 19K is functionally similar to this EGFR domain, we will construct a chimeric EGFR in which the acidic domain is replaced with the corresponding domain of 19K. The role of the chimeric receptor in calcium mobilization and ligand-dependent internalization will be determined. The proposed studies should facilitate understanding of the biochemical mechanism of 19K-mediated transformation.